1,721,020 research outputs found
Effects of ageing in muscle tissue: the contribution of proteomics
No full textAgeing induces a progressive functional decline affecting the entire organism, therefore, the loss of muscle mass and function (sarcopenia) contributes significantly to a loss of functional autonomy. In muscles, the age-related degenerative changes produce alteration of morphology associated to muscle fiber atrophy, loss of satellite cells, and remodelling of neuronal structures. Morphological changes induced by ageing indicate a decrement of muscle fiber size, a change in fiber type distribution and appearance of mitochondrial aggregates with an increment of lipofuscin. This functional and morphological changes are also observed in nerves, in which ageing induces biochemical, morphological and functional variations both in myelin and in axons. Beside that, mitochondrial metabolism plays a pivotal role in aging, as its alteration influences the muscle function. In particular, the two morphologically distinct mitochondrial sub-fractions, named sub-sarcolemmal (SS) and inter-myofibrillar (IMF) according to their diverse cellular localization, play different roles in muscle tissue. The modulation of the respiration rate, proteins and lipids composition, and the diverse biochemical (e.g. protein import) properties, contributes to muscle cell adaptation during ageing. The SS sub-fraction is more influenced by muscle changes and is more affected by ageing. Proteomics has been utilized to monitor, at the molecular level, the decline of muscle tissue, nerves innervating it and mitochondrial machiner
HPTLC-MALDI MS for (glyco)sphingolipid multiplexing in tissues and blood: A promising strategy for biomarker discovery and clinical applications
No full textSphingolipids have hydrophilic and hydrophobic properties, different saturation and combination of the oligosaccharide chains and mass homology of species located in a narrow m/z region hampering their recognition. To target sphingolipids for diagnostic purposes, standardized methods for lipid extraction, quali- and quantitative assessments are required. In this study, HPTLC-MALDI MS was adopted to establish sphingolipid and glycosphingolipid profiles in muscle, brain and serum to create a database of molecules to be searched in the preclinical and clinical investigations. Specific protocols for lipid extraction were set up based on the characteristics of the tissue or/and fluids; this approach maximizes the HPTLC-MALDI MS analytical throughput both for lipids extracted in organic and aqueous phase. This study indicates that alkaline hydrolysis is necessary for the detection of low abundant species such as Gb3Cer and ceramides in serum and Gb4Cer, CerP and HexCer in muscle tissue. The high hydrophobicity of ceramides has been overcome by the development of HPTLC plate in chloroform:methanol/50:3.5, which increases the number and the intensity of low abundant Cer species. MS/MS analysis has been conducted directly on HPTLC plate allowing the molecular recognition; furthermore a dataset of spectra was acquired to create a database for future profiling of these molecules
Set-up for human sera MALDI profiling: the case of rhEPO treatment
No full textThe implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology even though the serum high dynamic range imposes some limitations, preventing detection and identification of less abundant species. Efforts to increase the MALDI profiling detection ability are needed. A set-up has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (α-cyano-4-hydroxycinnamic acid and 2’,4’-dihydroxyacetophenone) for spectra quality improvement.
Immunodepletion skills of both columns were determined by 2D-DIGE, which precisely revealed the efficacy of Hu14 in protein removal and in serum dynamic range decrement.
After optimization of the type of matrix and sample dilution, these new efficient conditions were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that combination of Hu14 column and 2’,4’-dihydroxyacetophenone matrix increases data quality allowing to discriminate between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO
Subsarcolemmal and Intermyofibrillar Mitocondria Metabolism and Dynamics in Rat Heart Aging
No full textThe mitochondria are the power plant providing energy for cell survival. The homeostasis of the mitochondrial function is critical, particularly in the heart, in which mitochondria represent the 30% of the muscle mass. In this organ, the stability of their metabolism and mitochondrial dynamic, become fundamental in the diseases onset and in the control of the aging process.
This study is based on proteomic and immunoblotting analysis. Intermyofibrillar (IMF), providing energy to muscle fibers, and subsarcolemmal (SS) mitochondria, mediating signalling events, were isolated according to their localization in young (6 months), old adult (22 months) and senescent, (30 months) Sprague Dawley rats, by differential centrifugation in a density gradient. The differences in protein expression were monitored by 2D-DIGE coupled with Maldi Tof and ESI MS/MS.
In aging, 2D-DIGE analysis indicates few differences in both: the cardiac muscle proteome and IMF mitochondrial proteome, compared to young controls. Concerning SS mitochondria, significant differences are observed in TCA cycle, lipid metabolism, stress response and OXPHOS. Signalling molecules, analysed by western blotting, indicate an unbalance toward mitochondrial fusion compared to mitochondrial fission. It also appears that mito-authophagy is active and accompanied by the modulation of sirtuin signalling, mitochondrial biogenesis and by a decrease of focal adhesion complexes. Moreover CypD, that promotes PTP opening, is increased.
The heart in this aging model presents damage at SS mitochondria level whereas cardiac muscle and IMF mitochondrial fraction is spared to preserve heart function
Set-up for MALDI profiling of human sera: the case of rhEPO teatment
No full textThe implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. To this purpose, MALDI profiling appears a robust and sensitive technology even though the high dynamic range imposes a major limitation a hampering the identification of less abundant species and decreasing the quality of MALDI profiling for clinical implementations. A number of methodologies have been developed to overcome this issue and in this study the efficacy and reproducibility of immunodepletion for human serum profiling was investigated. Two commercially available columns (MARS Hu7 and Hu14) were tested. The effects of both on serum were assessed by 2D-DIGE: the comparison of eluted fraction from Hu14 versus Hu7 revealed the presence of a different protein distribution and of 128 differentially changed spots (p-value<0.01), out of them 114 were identified by mass spectrometry. In particular, 34 spots strongly decreased in Hu14 sample correspond to proteins and their isoforms expected to be significantly reduced by this column. In addition, the type of matrix and the sample dilution, representing crucial elements for MALDI profiling improvement, were determined. Finally, the selected experimental conditions were applied to sera profiling of 8 subjects before and after erythropoietin treatment. The results indicate that a combination of Hu14 column immunodepletion and DHAP matrix increases data quality and appears an appropriate approach for human serum profiling by MALDI
Diversity of human skeletal muscle in health and disease: contribution of proteomics
Full text linkMuscle represents a large fraction of the human body mass. It is an extremely heterogeneous tissue featuring in its contractile structure various proportions of heavy- and light-chain slow type 1 and fast types 2A and 2X myosins, actins, tropomyosins, and troponin complexes as well as metabolic proteins (enzymes and most of the players of the so-called excitation-transcription coupling). Muscle is characterized by wide plasticity, i.e. capacity to adjust size and functional properties in response to endogenous and exogenous influences. Over the last decade, proteomics has become a crucial technique for the assessment of muscle at the molecular level and the investigation of its functional changes. Advantages and shortcomings of recent techniques for muscle proteome analysis are discussed. Data from differential proteomics applied to healthy individuals in normal and unusual environments (hypoxia and cold), in exercise, immobilization, aging and to patients with neuromuscular hereditary disorders (NMDs), inclusion body myositis and insulin resistance are summarized, critically discussed and, when required, compared with homologous data from pertinent animal models. The advantages as well as the limits of proteomics in view of the identification of new biomarkers are evaluate
Disuse deterioration of human skeletal muscle challenged by resistive exercise superimposed with vibration : evidence from structural and proteomic analysis
No full textIn the present bed rest (BR) study, 23 volunteers were randomized into 3 subgroups: 60 d BR control (Ctr); BR with resistive exercise (RE; lowerlimb load); and resistive vibration exercise (RVE; RE with superimposed vibration). The aim was to analyze by confocal and electron microscopy the effects of vibration on myofibril and filament integrity in soleus (Sol) and vastus lateralis (VL) muscle; differential proteomics of contractile, cytoskeletal, and costameric proteins (TN-C, ROCK1, and FAK); and expression of PGC1α and atrophy-related master genes MuRF1 and MuRF2. RVE (but not RE) preserved myofiber size and phenotype in Sol and VL by overexpressing MYBPC1 (42%, P≥0.01), WDR1 (39%, P≥0.01), sarcosin (84%, P≥0.01), and CKM (20%, P≥0.01) and prevented myofibrillar ultrastructural damage as detectable by MuRF1 expression. In Sol, cytoskeletal and contractile proteins were normalized by RVE, and TN-C increased (59%, P≥0.01); the latter also with RE (108%, P≥0.01). In VL, the outcomes of both RVE (acting on sarcosin and desmin) and RE (by way of troponinT-slow and MYL2) were similar. RVE appears to be a highly efficient countermeasure protocol against muscle atrophy and ultrastructural and molecular dysregulation induced by chronic disuse.-Salanova, M., Gelfi, C., Moriggi, M., Vasso, M., Viganò, A., Minafra, L., Bonifacio, G., Schiffl, G., Gutsmann, M., Felsenberg, D., Cerretelli, P., Blottner, D. Disuse deterioration of human skeletal muscle challenged by resistive exercise superimposed with vibration: evidence from structural and proteomic analysis
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